Assessment of smell disturbances 6 months after COVID-19 in Polish population

Considering the frequency and severity of olfactory disorders associated with SARS-CoV-2 infection, attention to the olfactory loss has expanded. The aim of our study was to assess of smell disturbances 6 months after COVID-19. The study population consisted of 2 groups: 196 Post-COVID-19 patients who were hospitalized because of COVID-19, control sample–130 patients without reported smell disorders from general population-Bialystok PLUS study. People from both groups were asked to participate in the Sniffin Sticks Test (half year after the disease). Sniffin Sticks Test consisted of 12 standardized smell samples. The participant's test score was counted based on correct scent recognition. Middle/older age was related with lower likelihood of olfaction recovery. The biggest differences in recognition of particular fragrances were observed for: orange and lemon, lemon and coffee (p.adj < 0.001). Patients had the greatest problem in assessing smell of lemon. The comparison of scores between Delta, Omicron, Wild Type, Wild Type Alpha waves showed statistically significant difference between Delta and Wild Type waves (p = 0.006). Duration of the disease (r = 0.218), age (r = -0.253), IL-6 (r = -0.281) showed significant negative correlations with the score. Statistically significant variables in the case of smell disorders were Omicron wave (CI = 0.045–0.902; P = 0.046) and Wild Type wave (CI = 0.135–0.716; P = 0.007) compared to Delta wave reference. Moreover, patients with PLT count below 150 000/μl had greater olfactory disorders than those with PLT count over 150 000/μl. There are: smell differences between post-COVID-19 patients and healthy population; statistically significant difference between Delta and Wild Type waves in Post-COVID-19 group in score of the Sniffin Sticks Test. Smell disturbances depend on the age, cognitive impairments, clinical characteristics of the COVID-19 disease and sex of the patient.

The coronavirus disease 2019 (COVID-19) pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 .Moreover, the first cases of this disease were reported in Wuhan, Hubei Province, China 2 .SARS-CoV-2 is usually associated with pulmonary infection which leads to pneumonia, but recent studies demonstrate that other organs may be affected e.g., in the gastrointestinal, cardiovascular, nervous, and immune systems 3,4 .
An olfactory loss after viral infection is well-documented and the viruses responsible for this condition are adenovirus, influenza virus, rhinovirus, and coronavirus 5,6 .Considering the frequency and severity of olfactory impairment connected with SARS-CoV-2 infection, attention to the olfactory loss after viral illness has expanded significantly due to the COVID-19 pandemic.Furthermore, SARS-CoV-2 associated olfactory dysfunction is rarely connected with the nasal obstruction or rhinorrhea compared to other respiratory viruses causing olfactory loss 7 .The incidence of acute olfactory loss in acute phase of COVID-19 ranges from 34 to 86% [8][9][10] .After 2 weeks of recovery following COVID-19, an estimated 44% to 64% of these individuals recover olfaction 7,10 .In addition, some studies described recovery from initial COVID-19 for up to half year [11][12][13][14][15][16] .For example, Petrocelli et al.  studied that 6 months after the onset of the disease, approximately 6% of patients still have a severe persistent olfactory disorders.The functional recovery is most common in the first two months in relation to smell and after this time, the likelihood of improvement is significantly decreased 11 .Some theories have been described to explain the pathogenesis of COVID-19-associated anosmia, including oedema of the olfactory cleft mucous membrane, damage of olfactory epithelium either within the olfactory receptor cells or the supporting non-neural cells, damage to the olfactory bulb, and impairment of the central olfactory pathways 17 .Unfortunately, the pathogenesis of COVID-19-associated anosmia is still not fully explained.It seems to be due to sensorineural damage, with infection of the olfactory epithelium support cells via the angiotensin-converting enzyme 1 receptor and disorder of the olfactory epithelium caused by inflammatory process, and probably with direct olfactory sensory neurons infection mediated by the neuropilin-1 receptor 17 .It might also be related to genetic variables, involvement of the higher olfactory pathways and a conductive component of olfactory disorders 17 .
In our study we aimed to assess the smell disturbances six months after COVID-19 using Sniffin Sticks Test.

Material and methods
The study population consisted of 2 groups The Bialystok PLUS study describes the health of the local community by analysis of the examinations and questionnaires of a carefully selected cohort representative for the local population.It is being conducted from 2018 on a sample of randomly selected Bialystok residents aged 20-80 years old 18 .
Patients were selected according to sex, age and wave number.Patients were selected based on anti-N negative results.
We assessed the patients 6 months after the infection after each wave of pandemics.Particular variant of the virus was assessed by PCR method.The variant PCR testing was performed in the local population and it was described in: "RT-COVAR map: Monitoring of SARS-COV-2 variants and mutations in Poland" 19 20 .
People from both groups were asked to participate in the Sniffin Sticks Test.Sniffin Sticks Test is an examination used to assess olfactory disorders.This test consists of 12 reusable standardized fragrance samples (food and non-food smells).The fragrances in our study's samples are: 1. Orange; 2. Leather; 3. Cinnamon; 4. Mint; 5. Banana; 6. Lemon; 7. Licorice; 8. Coffee; 9. Cloves; 10.Pineapple; 11.Rose; 12. Fish.For the test, sticks with a material soaked in a fragrance are used (Fig. 1).After removing the cap, the tip of the stick is placed in front of the participant's nostrils.The proband must not touch his nose to the stick.The Sniffin Sticks come with 12 answer cards.There are 4 answers on each card.The participant chooses one scent from the list presented to him.The person conducting the test writes whether the participant correctly recognized the smell.The participant's test score was counted based on correct scent recognition and could range from 0 to 12.

Statistical methods
Statistical analyses were performed using the R programming language 21 .All necessary data transformations were performed using "tidyverse" package 22 , "ggstatsplot" package for visualizations of statistical tests 23 .Statistical significance was determined using a significance level of α = 0.05, where a p-value below this threshold was considered important.To compare the differences, the Kruskal-Wallis test was utilized.In addition, a post-hoc analysis was conducted using the Dunn test to perform pairwise comparisons among multiple groups.The resulting p-values were adjusted using the Bonferroni correction, and significant differences were identified if the adjusted p-value was below 0.05.In addition to group comparisons, the relationship between continuous variables was examined using Pearson's correlation coefficient and Dunn test for relationship between continuous variables and factors.Quasi-Poisson and Logistic Regression models were used for statistical inference.Propensity score weighting using optimal full weighting was used to mitigate the difference in age between Group I and Group II.Propensity scores were estimated using a logistic regression of the treatment on the covariates.Age was used as the only covariate.Average effect of the treatment (ATT) was the target estimand.After weighting, all standardized mean differences for the covariates were below 0.01, indicating adequate balance.Full weighting uses all treated and all control units, so no units were discarded.

Ethical approval statement
This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Bioethical Committee of Medical University of Bialystok (Poland) on 26 November 2020 (approval number: APK.002.346.2020).

Informed consent statement
Informed consent was obtained from all subjects involved in the study.

Baseline characteristics
In Group I the mean age was 53.7 years (standard deviation = 12.6).51% individuals were male, 49% were female.The mean age of the patients in Group II was 64.9 years (standard deviation = 12.8).In terms of sex distribution in control sample, 55% individuals were male and 45% individuals were female.

Olfactory function according to clinical outcomes
Propensity score weights estimated with logistic regression were used to mitigate the difference in age between Group I and Group II.The results are presented in Table 1.

Sniffin Sticks Test results for control sample and research sample
196 patients from Group I received twelve fragrance samples which were the Sniffin Sticks Test.The fragrances in our study's samples are (in that order): 1. Orange; 2. Leather; 3. Cinnamon; 4. Mint; 5. Banana; 6. Lemon; 7.  www.nature.com/scientificreports/ was correctly distinguished by 113 patients (x̅ = 0.87 ± 0.34).Twelfth sample (fish) was correctly recognized by 118 of the 130 individuals (x̅ = 0.91 ± 0.29) (Fig. 2).In addition, the results of correctly recognized fragrances in research sample depending on the period of the COVID-19 pandemic in which the patient was ill are summarized in Fig. 3.

Comparison of total score of the Sniffin Sticks Test between different waves in research sample
It was also decided to make a comparison of total score of the Sniffin Sticks Test between Delta, Omicron, Wild Type and Wild Type Alpha waves.The particular waves consisted of: 87 people in Delta wave, 11 people in Omicron wave, 72 people in Wild Type wave and 26 people in Wild Type Alpha wave.Dunn's test with Bonferroni correction was used for this comparison.Only the difference between the Delta wave and the Wild Type wave turned out to be statistically significant.The results are shown in Fig. 4.

Differences in recognizing particular smells in Group I
After assessment recognition of fragrances from the Sniffin Sticks data, differences in recognition of individual smells were compared.Dunn's test with Bonferroni correction was used for this comparison.Only statistically significant data (p-value < 0.05) were used for the comparison and differences in recognition of individual smells were confirmed.The greatest differences were observed in the comparison of the scents of orange and lemon (p.adj < 0.001; Z statistic = -15.313),lemon and coffee (p.adj < 0.001; Z statistic = 14.659), fish and lemon (p.adj < 0.001; Z statistic = 14.266), mint and lemon (p.adj < 0.001; Z statistic = 14.004) and rose and lemon (p.adj < 0.001; Z statistic = -10.069).Other statistically significant differences in the recognition of individual smells are demonstrated in Table 2.

Correlation of total score of the Sniffin Sticks Test with various clinical data in Group I and Group II
During the Sniffin Sticks Test, a lot of clinical and laboratory data were evaluated, which were used to create a correlation of total score of the Sniffin Sticks Test with these clinical data.The Pearson correlation was used for this statistics.Statistically significant variables for control sample are summarized in Table 3 and for research sample in Table 4.
Moreover, qualitative variables evaluated during the Sniffin Sticks Test required statistical assessment in the Dunn test.Only the categorical variables of the research sample (Group I) turned out to be statistically significant and are presented in Table 5.

Impact of various predictors on olfactory disorders during COVID-19 and on the result of the Sniffin Sticks Test in Group I
It was also decided to make models explaining the impact of various predictors on smell disorders during disease and on the result of the Sniffin Sticks Test.It turned out that the only statistically significant predictors in the  6 and logistic regression was used to demonstrate them (Table 6).
It is also worth noticing that in the case of the Quasi-Poisson Regression model (Table 7), the only statistically significant variables were sex (Incidence Rate Ratio = 1.068;Standard error = 0.029; Confidence interval = 1.012 -1.127; P value = 0.016) and age (Incidence Rate Ratio = 0.996; Standard error = 0.029; Confidence interval = 0.993 -0.998; P value < 0.001) in correlation with the dependent variable-the result of the Sniffin Sticks Test.

Assessment of the severity of COVID-19 infection in Group I
Analysis of associations between COVID-19 severity assessed on laboratory parameters (IL-6 > 100 pg/ml, D-dimer concentration over 1000 ng/ml and PLT count below 150 000/μl) and persistence of smell loss showed statistically significant differences when PLT was examined 24 .Patients with PLT count below 150 000/μl had greater olfactory disorders than those with PLT count over 150 000/μl.The relation of smell disorders with severity of COVID-19 classified based on laboratory criteria abnormalities during COVID-19 is presented on Fig. 5.

Discussion
Olfactory impairment is connected with lower quality of life, depression, diminished food satisfaction, inability to recognize dangerous environmental hazards, and decreased social well-being 25,26 .It has also been associated with increased mortality in elderly people 27 .
The mean age of the patients in research sample who participated in our study was 53.7 ± 12.6 years.These results are consistent with previous research indicating a connection between persistent olfactory loss and middle and older age 11,14 .COVID-19-related smell disorders rarely occur in either extreme age and are most popular in the 40-50-year-old age bracket 28,29 .There is correlation between age category and volume of expression of angiotensin-converting enzyme 2 (ACE2) receptors as well as other entry proteins or sustentacular cells 28,29 .For the middle-age group, the greatest volume of ACE2 expression was demonstrated, and nasal gene expression of ACE2 was found to raise with age (between 4 and 60 years old) 28,29 .
The nasal cavity plays an essential role in COVID-19 because it is an area of viral replication and one of the entry ways for SARS-CoV-2.SARS-CoV-2 appears to have its own ways of aggression to the nasal neuroepithelium, with a preference for neural implication over the mucous membrane in the nose 30,31 .Olfactory impairment in COVID-19 patients has been studied in research that assessed the symptom subjectively 10,32,33 .In COVID-19, the prevalence of self-reported smell impairment varies greatly between those studies, ranging from 23.7% to nearly 90% 9,[31][32][33] .Subjective techniques of evaluation, on the other hand, are prone to a variety of biases and result in significant inaccuracy 34,35 .Alpha variants and Delta variants 39 .Furthermore, Omicron variants might be associated with a lower risk of developing long-COVID syndrome 39 .This was confirmed by Boscolo-Rizzo et al. which showed the prevalence and the severity of smell and taste disorders after COVID-19 has dropped significantly with the advent of the Omicron variant 40 .The most striking difference was observed for loss of sense of smell, a pathognomonic feature of earlier waves of SARS-CoV-2 infection, in Omicron wave presented in less than 20% of cases 41 .Interestingly, the two symptoms that were consistently more prevalent among Omicron than among Delta cases (regardless of vaccination status) were sore throat and hoarse voice 42 .People with smell impairment may compensate by relying on other senses and recalling cognitive memories of particular fragrances 43 .It is expected that COVID-19 would cause chronic smell impairment in hundreds of thousands of people 44 .This research gives helpful data about short-term olfactory loss, allowing physicians to provide correct anticipatory guidance to patients.In our opinion, future research should focus on testing modifications to existing ways of treatment as well as the development of new therapies for smell disorders.
Interestingly, Petrocelli et al. observed a two-month asymptote after which there was little recovery.They proposed that the two-month period is a temporal threshold at which it was sensible to start empiric treatment 11 .Therefore, we think that the limitation of our study is the absence of performance of the Sniffin Sticks Test also during the disease and two months after the disease to compare the results at the same timepoint.Furthermore, other limitations are: the lack of measures of phantosmia and parosmia, which are main components of COVID-19 smell impairment, and the high number of subjective factors in this study so our results cannot be directly generalized to all individuals with a post-COVID olfactory disorder and should be verified in a larger prospective cohort study.However, our results showed, that patients after COVID-19 had higher tendency to smell disturbances (which may be a sign of neurodegeneration of olfactory nerves), even if patients were relatively younger.
To sum up, in the present study, we assessed smell disturbances 6 months after COVID-19 in Polish population.A large majority of patients recovered olfactory function within six months of COVID-19 onset assessed by Sniffin Sticks Test.The findings of this research might be used to inform individuals about the chance of recovery of smell after COVID-19 and estimation of predictors of smell disturbances after the COVID-19.

Conclusions
1.There are smell differences between post-COVID-19 patients and healthy population.

Figure 3 .
Figure 3. Graph comparing correctly and incorrectly recognized fragrances in research sample depending on the COVID-19 wave in which the patient was ill.Frequency shown as labels on bars.

Figure 4 .
Figure 4. Comparison of total score of the Sniffin Sticks Test between Delta, Omicron, Wild Type and Wild Type Alpha waves in Group I.

2 .
There was statistically significant difference between the Delta wave and the Wild Type wave in Post-COVID-19 group in the total score of the Sniffin Sticks Test.

Figure 5 .
Figure 5. Assessment of the severity of COVID-19 infection in Group I.

Table 1 .
Regression model of Sniffin Sticks Test total score in Group I and Group II.Ordinary least squares regression.

Table 4 .
Correlation of the mean of total points of the Sniffin Sticks Test of Group I with various clinical data.

Table 5 .
Correlation of the dependent variable (Total score of the Sniffin Sticks Test) with the independent variables (sex, use of Dexaven and use of Ceftriaxone) in Group I.

Table 6 .
Logistic regression model of smell disorders during COVID-19 in Group I.

Table 7 .
Quasi-Poisson model of Sniffin Sticks Test total score in Group I.